Molecular mechanisms by which primate lentiviruses, such as HIV-1, are sexually transmitted, have yet to be fully elucidated. It has been demonstrated in our laboratories that endogenous reverse transcription of lentiviruses can occur within the intact virion, before infection of target cells. This is a biochemically-active process, a new stage in the retroviral life-cycle, and is altered by the microenvironment to which HIV-1 or SIV virions are subjected. Stimulation of endogenous reverse transcription within virion particles, without non-physiological permeabilization of the viral envelope, has been entitled "natural endogenous reverse transcription" (NERT). This molecular mechanism has been shown to stimulate HIV-1 infection in initially-quiescent PBMCs, as well as non-proliferating macrophages. As such, this process may be important in augmenting the sexual transmission of HIV-1, as certain genital secretions have been shown to stimulate NERT within HIV-1 virion particles. We have also demonstrated that triphosphorylated derivatives of nucleoside-analogue reverse transcriptase inhibitors (NRTIs) and non-nucleoside-analogue RT inhibitors (NNRTIs) can directly enter lentivirions via pores in the virion particles' surface, and decrease viral infectivity by inhibiting NERT. In this proposal, we will directly investigate intravirion reverse transcription as a target for molecular virucides. In the first specific aim, the ability to develop "molecular virucides" will be explored, targeting intravirion reverse transcription. In vitro analyses of SIV/HIV-1 and SHIV-RT constructs will be performed using inhibitory agents of intravirion reverse transcription with NNRTIs (for HIV-1 and SHIV-RT) and triphosphorylated NRTIs (for HIV-1, SIV, and SHIV-RT). Studies will also include analysis of changes in intravirion reverse transcription, intracellular reverse transcription, and viral infectivity. Agents showing promise will then be properly pre-formulated, with toxicity analyses, and formulated for in vivo utility as virucidal preparations and re-analyzed for antiviral activity in specific aim II. The proposed experiments will set the groundwork for future development of a macaque transmission model to analyze this virucide approach in vivo. These studies are also designed to move analysis of intravirion reverse transcription from the bench towards clinical utility in altering sexual transmission of human lentiviruses.